Macrophages/microglia in neuro-inflammation associated with neurodegenerative diseases

ABSTRACT

Described herein are methods of treating neuron inflammation conditions, for example, Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, ischemic stroke, and prion disease, comprising administering a therapeutically effective amount of cromolyn or a cromolyn derivative compound.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/382,192, filed Aug. 31, 2016, which ishereby incorporated herein by reference in its entirety.

FIELD

The invention encompasses methods of treating a neuron inflammationcondition comprising administered a therapeutically effective amount toa patient in need thereof of at least one compound having the followingformula:

BACKGROUND

Strategies to modulate monocyte and microglial activity have beenstudied, especially those that can protect against microglia-mediatedneurotoxicity. (See, Zhao et al., “Protective effects of ananti-inflammatory cytokine, interleukin-4, on motoneuron toxicityinduced by activated microglia,” J. Neurochem. 2006, 99:1176-1187;Heneka et al., “NLRP3 is activated in Alzheimer's disease andcontributes to pathology in APP/PS1 mice,” Nature, 2013,493(7434):674-8; Theeriault, et al., “The dynamics of monocytes andmicroglia in Alzheimer's disease,” Alzheimers Res Ther., 2015, 7:41; Nauet al., “Strategies to increase the activity of microglia as efficientprotectors of the brain against infections,” Front Cell Neurosci., 2014,8:138.) Overall, it is clear that more focused studies are needed tobetter establish how each inflammatory state can modulate the pathologyof neurodegenerative diseases such as Alzheimer's Disease (AD) andAmyotrophic Lateral Sclerosis (ALS). Early activation of monocytes andmicroglia has potential to decelerate neurodegenerative progression bymodulating immune responses to increase the intrinsic phagocyticcapacity of monocytes and microglia without triggering secretion ofpro-inflammatory cytokines that could worsen neurodegeneration.

The role of the neuro-inflammatory response in the presence of amyloidplaques and neurofibrillary tangles in the brain and its associatedneuronal loss in the pathology of AD is well established and extensivelystudied. See, Walker et al., “Immune phenotypes of microglia in humanneurodegenerative disease: challenges to detecting microglialpolarization in human brains,” Alzheimers Res Ther., 2015, 7:56;Theerialut et al., 2015; Wilcock, D M, “A Changing Perspective on theRole of Neuroinflammation in Alzheimer's Disease,” International Journalof Alzheimer's Disease, 2012, Article ID 495243; McGeer et al.,“Targeting microglia for the treatment of Alzheimer's disease,” ExpertOpin Ther Targets, 2015, 19(4):497-506). Numerous studies show thatmicroglial-mediated inflammation contributes to the progression of ADand that microglial cells are found in close association with amyloid-β(Aβ) deposits. (See, Mandrekar, et al., “Microglia and Inflammation inAlzheimer's Disease,” CNS Neurol Disord Drug Targets, 2010, 9(2):156-167).

It is known that the changes in properties of microglia—thebrain-resident macrophages—depend on their response to different stimuliin their microenvironment (e.g. cytokines), resulting in a range ofphenotypes. Based on the changes in expression of cytokines, receptors,and other markers, monocyte and macrophage states have been defined as:classical activation (M1), alternative activation (M2a), type IIalternative activation (M2b), and acquired deactivation (M2c). (See,Walker et al., 2015; Martinez et al., “Alternative activation ofmacrophages: an immunologic functional perspective,” Annu Rev Immunol.2009, 27:451-83; Mantovani et al., “The chemokine system in diverseforms of macrophage activation and polarization,” Trends Immunol., 2004,25:677-686; Sternberg, E M., “Neural regulation of innate immunity: acoordinated nonspecific host response to pathogens,” Nat Rev Immunol.,2006, 6(4):318-28). Recently, a number of studies have attempted toelucidate the role of these phenotypes in the AD brain and determine themechanisms through which these cells contribute to AD-relatedneuro-inflammation. (See, Mandrekar et al. 2012; McGeer et al., 2015;and Wilcock, 2012).

Interaction of microglia with fibrillar Aβ leads to their phenotypicactivation, and has recently been suggested to play a role inneuroprotection. (See Zhao et al., 2006; Figueiredo et al.,“Neuron-microglia crosstalk up-regulates neuronal FGF-2 expression whichmediates neuroprotection against excitotoxicity via JNK1/2,” J.Neurochem., 2008 October, 107(1):73-85). It has been shown in numerousstudies, in both mice and humans, that glial cells respond to thepresence of AD pathological lesions (plaques and tangles) by changingtheir morphological characteristics, expressing numerous cell surfacereceptors, and surrounding the lesions. (See, Perlmutter et al.,“Morphologic association between microglia and senile plaque amyloid inAlzheimer's disease,” Neurosci Lett., 1990, 119:1, 32-36; Combs, et al.,“Identification of microglial signal transduction pathways mediating aneurotoxic response to amyloidogenic fragments of β-amyloid and prionproteins,” J. Neurosci., 1999, 19:3, 928-939). On the other hand,macrophage and microglial activation in response to cellular debris inthe AD brain, and the subsequent release of pro-inflammatory cytokines,leads to accelerated neurodegeneration. This, in turn, creates morecellular debris and accelerates disease progression. (See, Rubio-Perezet al., “A Review: Inflammatory Process in Alzheimer's Disease, Role ofCytokines,” Scientific World Journal, 2012, 756357; McGeer, et al., “Theimportance of inflammatory mechanisms in Alzheimer disease,” Exp.Gerontol. 1998, 33:5, 371-378; Akiyama, et al., “Inflammation andAlzheimer's disease,” Neurobiol Aging, 2000, 21(3), 383-421; Liu, etal., “TLR2 is a primary receptor for Alzheimer's amyloid β peptide totrigger neuroinflammatory activation,” J. Immunol. 2012,188(3):1098-107).

Several studies have focused on microglial activation and its role inthe clearance of AD lesions leading to the reduction of amyloid depositsin the brain. (See, DiCarlo, et al., “Intrahippocampal LPS injectionsreduce Aβ load in APP+PS1 transgenic mice,” Neurobiol of Aging, 2001,22:6, 1007-1012; Herber, et al., “Time-dependent reduction in Aβ levelsafter intracranial LPS administration in APP transgenic mice,” Exp.Neurol., 2004, 190(1):245-53; Liu, et al., 2012). While residentmicroglial cells surrounding Aβ plaques are not as efficacious indegrading Aβ as newly infiltrated macrophages or monocytes (See,Thériault, et al., 2015; Varnum, et al., “The classification ofmicroglial activation phenotypes on neurodegeneration and regenerationin Alzheimer's disease brain,” Arch. Immunol. Ther. Exp. (Warsz), 2012,60(4):251-66), it has been shown that microglia are indeed capable ofinternalizing fibrillar and soluble Aβ, but are unable to process thesepeptides. (See Chung, et al., “Uptake, degradation, and release offibrillar and soluble forms of Alzheimer's amyloid beta-peptide bymicroglial cells,” J Biol. Chem., 1999, 274:32301-8).

Further, it has been postulated that microglia undergo a switch from anM2- to an M1-skewed activation phenotype during aging. (See, Heneka etal., 2013; Varnum, et al., 2012; Gratchev, et al., “Mphi1 and Mphi2 canbe re-polarized by Th2 or Th1 cytokines, respectively, and respond toexogenous danger signals,” Immunobiology, 2006, 211(6-8):473-486;Colton, et al., “Expression profiles for macrophage alternativeactivation genes in AD and in mouse models of AD,” J. Neuroinflammation,2006, 3:27). However, how the immune response in the brain is driven inAD is still a matter of debate, especially whether neuroinflammation canbe triggered by age-related systemic inflammation. (See, Thériault, etal., 2015). It has been shown that stimulation of microglia couldenhance their intrinsic phagocytic capacity to degrade Aβ moreefficaciously; a number of strategies to modulate microglial responsehave been proposed. (See, Mandrekar, 2010; Kiyota, et al., “CNSexpression of anti-inflammatory cytokine interleukin-4 attenuatesAlzheimer's disease-like pathogenesis in APP+PS1 bigenic mice,” FASEB J.2010, 24:3093-3102; He, et al., “Deletion of tumor necrosis factor deathreceptor inhibits amyloid beta generation and prevents learning andmemory deficits in Alzheimer's mice,” J. Cell Biol., 2007, 178:829-841;Varnum, et al., 2012).

It has been shown that microglia are activated by extracellularlydeposited Aβ peptide (Lotz, et al., “Amyloid beta peptide 1-40 enhancesthe action of Toll-like receptor-2 and -4 agonists but antagonizesToll-like receptor-9-induced inflammation in primary mouse microglialcell cultures,” J. Neurochem., 2005, 94:289-298; Reed-Geaghan, et al.,“CD14 and toll-like receptors 2 and 4 are required for fibrillarAβ-stimulated microglial activation,” J. Neurosci., 2009,29:11982-11992). This is similar to microglial activation in response tothe presence of interferon-γ (IFNγ), tumor necrosis factor alpha (TNFα)from T cells, or antigen-presenting cells. M1 activated microglia canproduce reactive oxygen species and result in increased production ofpro-inflammatory cytokines such as TNFα and interleukin (IL)-1β.

The M1-type response of microglial cells has been shown to lower amyloidload but exacerbate neurofibrillary tangle pathology. Shaftel et al.(Shaftel, et al., “Sustained hippocampal IL-1β overexpression mediateschronic neuroinflammation and ameliorates Alzheimer plaque pathology,”J. Clin. Invest., 2007, 117(6):1595-604) have shown that IL-1βexpression may underlie a beneficial neuroinflammatory response in AD,and that IL-1β overexpression in the hippocampus of APP/PS1 transgenicmice results in decreased amyloid burden. The authors suggest thatIL-1β-mediated activation of microglia is the mechanism for thereductions in amyloid deposition. Further, Montgomery et al.(Montgomery, et al., “Ablation of TNF-RI/RII expression in Alzheimer'sdisease mice leads to an unexpected enhancement of pathology:implications for chronic pan-TNF-α suppressive therapeutic strategies inthe brain,” Am. J. Pathol., 2011, 179(4):2053-70) have shown that intactTNF-receptor signaling is critical for microglial-mediated uptake ofextracellular amyloid-peptide. While M1 inflammatory phenotypes appearto improve the amyloid pathology in numerous studies, induction of M1phenotypes in tau transgenic mice or cell culture results in theexacerbation of tau pathology. (See, Kitazawa, et al.,“Lipopolysaccharide-induced inflammation exacerbates tau pathology by acyclin-dependent kinase 5-mediated pathway in a transgenic model ofAlzheimer's disease,” J. Neurosci., 2005, 28; 25(39):8843-53.; Li, etal., “Interleukin-1 mediates pathological effects of microglia on tauphosphorylation and on synaptophysin synthesis in cortical neuronsthrough a p38-MAPK pathway,” J. Neurosci., 2003, 1; 23(5):1605-11).

Macrophage M2 activation is associated with mediators that are known tocontribute to the anti-inflammatory actions and reorganization ofextracellular matrix (Zhu, et al., “Acidic mammalian chitinase inasthmatic Th2 inflammation and IL-13 pathway activation”, Science, 2004,304(5677):1678-82; Walker, et al., 2015; Wilcock, et al., 2012).Microglia with M2a phenotypes have increased phagocytosis and producegrowth factors such as insulin-like growth factor-1 andanti-inflammatory cytokines such as IL-10. Stimulation of macrophages byIL-4 and/or IL-13 results in an M2a state, sometimes called awound-healing macrophage (Edwards, et al., “Biochemical and functionalcharacterization of three activated macrophage populations,” J. LeukocBiol., 2006, 80(6):1298-307) and it is generally characterized by lowproduction of pro-inflammatory cytokines (IL-1, TNF and IL-6). The M2aresponses are primarily observed in allergic responses, extracellularmatrix deposition, and remodeling.

M2b macrophages are unique in that they express high levels ofpro-inflammatory cytokines, characteristic of M1 activation, but alsoexpress high levels of the anti-inflammatory cytokine IL-10. (See, MoserD M., “The many faces of macrophage activation,” J. Leukoc Biol., 2003,73(2):209-12).

Finally, the M2c macrophage state is stimulated by IL-10 and issometimes referred to as a regulatory macrophage. M2c macrophages haveanti-inflammatory activity that plays a role in the phagocytosis ofcellular debris without the classical pro-inflammatory response (See,Moser D M., 2003). These cells express TGFβ and high IL-10 as well asmatrix proteins. (See, Mantovani, et al., “The chemokine system indiverse forms of macrophage activation and polarization,” TrendsImmunol., 2004, 25:677-686; Wilcock, et al., 2012). Plunkett et al.(Plunkett, et al., “Effects of interleukin-10 (IL-10) on pain behaviorand gene expression following excitotoxic spinal cord injury in therat,” Exp. Neurol., 2001; 168:144-154) reported that IL-10 mediatedanti-inflammatory responses including decreasing glial activation andproduction of pro-inflammatory cytokines.

However, the mechanism of M2 microglial activation and role it plays inAD and plaque pathology is still not well understood. (See, Mandrekar,et al., 2010). Further, a number of studies suggested that there is aswitch in microglial activation status in response to diseaseprogression (Colton, et al., 2006; Jimenez, et al., “Inflammatoryresponse in the hippocampus of PS1M146L/APP751SL mouse model ofAlzheimer's disease: age-dependent switch in the microglial phenotypefrom alternative to classic,” J. Neurosci., 2008, 28:11650-11661). Ithas been reported in animal studies that microglial activationphenotypes switch from M2 to M1 during disease progression (Jimenez, etal., 2008; Nolan, et al., “Role of interleukin-4 in regulation ofage-related inflammatory changes in the hippocampus,” J. Biol. Chem.,2005; 280:9354-9362; Maher, et al., “Downregulation of IL-4-inducedsignalling in hippocampus contributes to deficits in LTP in the agedrat,” Neurobiol. Aging, 2005, 26:717-728), suggesting an increasedclassical activation phenotype over the alternative phenotype with age.It is generally agreed that microglia activated by extracellularlydeposited Aβ protect neurons by triggeringanti-inflammatory/neurotrophic M2 activation and by clearing Aβ viaphagocytosis. This is a potential avenue for new therapeutic targets.(See, He, et al., 2007; Yamamoto, et al., “Interferon-gamma and tumornecrosis factor-alpha regulate amyloid-beta plaque deposition andbeta-secretase expression in Swedish mutant APP transgenic mice,” Am. J.Pathol., 2007, 170:680-692; Yamamoto, et al., “Cytokine-mediatedinhibition of fibrillar amyloid-beta peptide degradation by humanmononuclear phagocytes,” J. Immunol., 2008, 181:3877-3886).

Mantovani et al. (Mantovani, et al., 2004) studied the effect of IL-4 asan important modulator of M2a microglial activation. It has been shownthat gene delivery of IL-4 into APP+PS1 mice partially suppressed glialaccumulation in the hippocampus, directly enhanced neurogenesis,restored impaired spatial learning, and also reduced Aβ deposition(Kiyota, et al., 2010).

Yamamoto et al. (Yamamoto, et al., 2007, 2008) examinedmacrophage-mediated Aβ degradation using pro- and anti-inflammatorycytokines in primary cultured human monocyte-derived macrophages (MDM)and microglia. These studies showed that anti-inflammatory andregulatory cytokines lead to an increase in M2a or M2c activation andenhanced Aβ clearance. Kiyota et al. (Kiyota et al., 2011) have shownsustained expression of IL-4 reduced astro/microgliosis, amyloid-βpeptide (Aβ) oligomerization and deposition, and enhanced neurogenesis.

Several approaches have been proposed to modulate microglial activationas potential targets for AD treatment. (See, Thériault, et al., 2015;Cherry, et al., “Neuroinflammation and M2 microglia: the good, the bad,and the inflamed,” J. Neuroinflammation, 2014, 11:98; Mandrekar, et al.,2010; Vernum, et al., 2012). It has been suggested that use ofanti-inflammatory drugs, like non-steroidal anti-inflammatory drugs(NSAIDs), to halt the progression of AD could be suppressing bothpro-inflammatory and anti-inflammatory activation by endogenousmolecules, inactivating the beneficial effect of M2 microglial functionsand endogenous mechanisms of plaque clearance. (See, Wilcock, et al.,2012, Cherry, et al., 2014; Theeriault, et al., 2015).

Research has focused primarily on two areas: anti-inflammatory agents totemper toxic effect of pro-inflammatory cytokines; and convertingmicroglia from this M1 state to an M2 state in which the toxic effectsare reduced and their phagocytic activity toward Aβ is enhanced. It wassuggested (McGreer, et al., 2012) that potential treatments should beadministered early in the disease progression.

Strategies that modulate monocyte and microglial activity have beenstudied, especially those that can protect against microglia-mediatedneurotoxicity (Zhao, et al., 2006; Heneka, et al., 2013; Therlaut, etal., 2015; Nau, et al., 2014). Overall, it is clear that more focusedstudies need to be performed to better establish how each inflammatorystate can modulate the pathologies of AD. It is generally accepted thatearly activation of monocytes and microglia has potential to decelerateAD progression by modulating immune responses to increase the intrinsicphagocytic capacity of monocytes and microglia without triggeringsecretion of pro inflammatory cytokines that could worsen AD.

SUMMARY

In certain embodiments, the invention encompasses methods of treating aneuron inflammation condition comprising administering a therapeuticallyeffective amount to a patient in need thereof of at least one compoundhaving the following formula:

In other embodiments, the method uses the following compounds:

In yet other embodiments, the neuron inflammation condition is at leastone of ALS, AD, ischemic stroke, or prion disease. In one embodiment,the compounds may be administered intraperitoneally (IP) and/orintravenously (IV). The compounds may be administered at a dose betweenabout 1 mg and about 1000 mg per day. The method of administration maybe transdermally or by inhalation.

In another embodiment, the method is a method of treating ALS furthercomprising co-administering CD4+; siRNA; miRNA that ameliorate ALS;glial morphology modifier; SOD1 control; Riluzole; or another M1; M2conversion active drug that controls neuroinflammation.

In certain embodiments, the invention relates to any of the methodsdescribed herein, provided the compound is not cromolyn disodium. Incertain embodiments, the invention relates to any of the methodsdescribed herein, provided the compound is not cromolyn disodium,F-cromolyn disodium, ET-cromolyn, or F-ET-cromolyn when the neuroninflammation condition is AD.

In certain embodiments, the invention relates to any one of thefollowing compounds:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates the quantification of the plasmatic levels ofAβ_(x-40) and Aβ_(x-42) one week after treatment with PBS or escalatingdoses of Cromolyn Sodium (n=3-5 mice/group).

FIG. 1B illustrates representative images of localization of amyloiddeposits (6E10) and microglia (Iba1) in mice treated with CromolynSodium (3.15 mg/kg) or PBS daily for seven days. The bar figureillustrates the results from analyzing plaques for each animal. Scalebar=10 μm.

FIG. 1C illustrates the effect of Cromolyn Sodium on microglial Aβuptake in vitro, where after the incubation, the concentrations ofAβ_(x-40) (FIG. 1C left) Aβ_(x-42) (FIG. 1C, right) in media weremeasured using Aβ ELISA.

FIG. 2 illustrates the plaques and the microglial cells surroundingthose deposits in Tg-2576 mice of the study of Example 2. The figureshows representative pictures of amyloid deposits and Iba-1 positivemicroglia.

FIG. 3 illustrates the results of BV2 microglial cells treated withcromolyn, and with cromolyn and ibuprofen exhibit increased Aβ42 uptakelevels relative to BV2 microglia treated with the vehicle.

FIG. 4 illustrates the results of an Aβ aggregation inhibition assayusing various compounds described herein.

FIG. 5 graphically illustrates that Cromolyn significantly affects thelevels of brain TBS soluble Aβ and the ratios of Aβ (42:40).

FIG. 6A shows naïve BV2 microglial cells treated with DMSO (control) for16 h. Afterwards, cells were incubated with fluorescently-labeled Aβ42and DMSO or cromolyn sodium for 2 hours. After incubation, cells werelabeled with a plasma membrane dye (PM) and imaged.

FIG. 6B shows naïve BV2 microglial cells treated with DMSO (control) for16 h. Afterwards, cells were incubated with fluorescently-labeled Aβ42and DMSO or cromolyn sodium for 2 hours.

FIG. 6C showns naïve BV2 microglial cells treated with cromolyn sodium(500 μM) for 16 hours. Afterwards, cells were incubated withfluorescently-labeled Aβ42 and DMSO or cromolyn sodium for 2 hours.After incubation, cells were labeled with a plasma membrane dye (PM) andimaged.

FIG. 6D showns naïve BV2 microglial cells treated with cromolyn sodium(500 μM) for 16 hours. Afterwards, cells were incubated withfluorescently-labeled Aβ42 and DMSO or cromolyn sodium for 2 hours.

FIG. 7A graphically illustrates that cromolyn sodium promotes microglialAβ42 uptake. BV2 microglial cells were treated with DMSO or differentconcentrations of cromolyn sodium for 16 hours. Then, cells wereincubated with soluble untagged Aβ42 and DMSO or cromolyn sodium for 2hours, and collected for ELISA analysis. Both naïve BV2 andBV2-CD33^(WT) microglial cells treated with cromolyn sodium exhibitedincreased Aβ42 uptake levels in comparison to cells treated with thevehicle (DMSO).

FIG. 7B graphically illustrates that cromolyn sodium promotes microglialAβ42 uptake. BV2 cells stably expressing CD33 (BV2-CD33^(WT)) weretreated with DMSO or different concentrations of cromolyn sodium for 16hours. Then, cells were incubated with soluble untagged Aβ42 and DMSO orcromolyn sodium for 2 hours, and collected for ELISA analysis. Bothnaïve BV2 and BV2-CD33^(WT) microglial cells treated with cromolynsodium exhibited increased Aβ42 uptake levels in comparison to cellstreated with the vehicle (DMSO).

FIG. 8 graphically illustrates that compound C8 displays toxicity whentested at 100 μM or higher concentration in LDH assay. Naïve BV2microglial cells were treated with DMSO or cromolyn derivatives for 3hours at different concentrations. C1, C2, C5, C6, C7 and C8 were testedat 10, 50, 100 and 150 μM, while C3 and C4 were assessed at 5, 25, 50and 75 μM due to solubility limit in DMSO. Afterwards, cells wereincubated with soluble untagged Aβ42 peptide and DMSO or cromolynderivatives for 2 hours. At the end of the treatment, cell media wascollected and compound toxicity was assessed with the lactatedehydrogenase (LDH) assay. BV2 microglial cells treated with thecromolyn derivative C8 exhibited increased toxicity at 100 and 150 μM incomparison to cells treated with the vehicle (DMSO).

FIG. 9 graphically illustrates that compound C4 promotes Aβ42 uptake innaïve BV2 microglial cells. BV2 cells were treated with DMSO (vehicle)or cromolyn derivatives at different concentrations ranging from 5 to150 μM for 3 hours. Then, cells were incubated with soluble untaggedAβ42 and DMSO or cromolyn derivatives for additional 2 hours andcollected for ELISA analysis. BV2 microglial cells treated with thecromolyn derivative C4 at 75 μM exhibited significantly increased Aβ42uptake levels in comparison to cells treated with the vehicle.

FIG. 10 graphically illustrates that compound C4 promotes Aβ42 uptake inmicroglial BV2-CD33^(WT) cells. Microglial cells stably expressingCD33^(WT) were treated with DMSO as control or cromolyn derivatives (C1,C3-8) at different concentrations for 3 hours. Afterwards, cells wereincubated with DMSO or cromolyn derivatives in the presence of the Aβ42peptide for additional 2 hours. Cell lysates were analyzed forintracellular levels of Aβ42 using an Aβ42-specific ELISA kit. Treatmentwith the cromolyn derivative C4 at 75 μM led to increased uptake of Aβ42in BV2-CD33^(WT) cells in comparison to DMSO treatment and displayed adose-dependent effect at 50 μM.

FIG. 11 graphically illustrates that compound C4 promotes Aβ42 uptake inBV2-CD33^(WT) cells. BV2-CD33^(WT) cells were treated with DMSO(vehicle) or cromolyn derivatives (C1, C2, and C4-7) at differentconcentrations for 3 hours. Afterwards, cells were treated with DMSO orcromolyn derivatives and soluble Aβ42 peptide for 2 hours. Cell lysateswere analyzed using Aβ42-specific ELISA kit and intracellular Aβ42levels were quantified. The cromolyn derivative C4 effectively inducedAβ42 uptake at 50 and 75 μM in BV2-CD33^(WT) cells in comparison tocells treated with DMSO.

DETAILED DESCRIPTION

Ischemic stroke, Alzheimer's Disease (AD), Amyotrophic Lateral Sclerosis(ALS or Lou Gehrig's disease), Prion and other neurodegenerativedisorders are associated with microglial activation and mast cellmigration, as well as with monocytes and other cell types that produce abarrage of toxic cytokines and debris that enhance inflammation. Incertain embodiments, the invention encompasses anti-inflammatorycompounds to reduce the toxic effect of pro-inflammatory cytokines byconverting microglia from a pro-inflammatory M1 state to an M2 state inwhich the toxic effects are reduced and their phagocytic activity towardamyloidosis, tauopathies and other cytotoxic events is enhanced. Incertain embodiments, the invention also encompasses the use of thecompounds to affect therapy early in the disease process.

Many drugs used as anti-inflammatory agents showed no efficacy in theconversion of microglia from M1 to M2, nor do they enhance themodulation of microglia from M1 to M2. To the best of applicant'sknowledge, the compounds described herein are the only effective,non-cytokine (e.g. IL-10) compounds exhibiting M1-to-M2 activity. Thus,in certain embodiments, the invention encompasses the compounds and themethods of treating neuron inflammation conditions by administration ofa therapeutic effective amount of at least one of the compounds.

In certain embodiments, compounds of the invention include those havingthe following formula and their analogs and isomers:

In addition, X may include, but is not limited to, halides, andOCO(C₁-C₈ alkyls). Alkyl groups include, but are not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and pentyl.Halides include fluoro, chloro, bromo, and iodo. Y may include, but isnot limited to, —CH₂OH, —CH₂OAc, or —CH₂OMe. Preferably, the compoundsof the invention include those compounds attached at the 5 position.

Specific compounds with the scope of the invention include:

In certain embodiments, compounds also include5-[3-(2-carboxy-4-oxochromen-5-yl)oxy-2-hydroxypropoxy]-4-oxochromene-2-carboxylicacid derivatives and isomeric forms.

In certain embodiments, the invention encompasses methods of treating avariety of neuron inflammation conditions. Neuron inflammationconditions include, but are not limited to, diseases such as ALS, autismspectrum disorder (ASD), ischemic stroke, and prion disease. In certainembodiments, the compounds may be used to treat ALS including, but notlimited to, slowing down or halting the progression of the disease. Incertain embodiments, the compounds may be administered in combinationwith other anti-inflammatory agents to control the spread of theprogressive and fatal effect of ALS.

In certain embodiments, the invention encompasses a combinationtreatment for ALS of M1, M2 conversion active drugs that controlneuroinflammation, such as the drugs in the above formulas, with otherimmune targeting therapies such as CD4+, siRNA, miRNA that amelioratesALS, glial morphology modifiers, SOD1 controls, or Riluzole, the onlyapproved drug for ALS.

In other embodiments, the compounds will slow down or halt neuron damagefor neurons located in the brain stem and/or the spinal cord, neurons,or motor neurons that affect voluntary body muscles.

In certain embodiments, the compounds may be administered using knownmethods for the administration of drugs, for example, IP, IV,transdermally, by inhalation. In certain embodiments, the inventionrelates to methods of treating or slowing down the aggressiveprogression of a neurological disease, such as AD, Ischemic Stroke, ALS,or Prion, and the compound is administered by infusion orintraperitoneal administration.

In certain embodiments, the invention also provides pharmaceuticalcompositions comprising one or more compounds described herein inassociation with a pharmaceutically acceptable carrier. Preferably thesecompositions are in unit dosage forms such as tablets, pills, capsules,powders, granules, sterile parenteral solutions or suspensions, meteredaerosol or liquid sprays, drops, ampoules, auto-injector devices orsuppositories; for oral, parenteral, intranasal, sublingual or rectaladministration, or for administration by inhalation or insufflation. Itis also envisioned that the compounds may be incorporated intotransdermal patches designed to deliver the appropriate amount of thedrug in a continuous fashion.

For preparing solid compositions such as powders and tablets, theprincipal active ingredient is mixed with a pharmaceutically acceptablecarrier, e.g. conventional tableting ingredients such as corn starch,lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,dicalcium phosphate or gums, and other pharmaceutical diluents, e.g.water, to form a solid preformulation composition containing ahomogeneous mixture. When referring to these preformulation compositionsas homogeneous, it is meant that the active ingredient is dispersedevenly throughout the composition so that the composition may be easilysubdivided into equally effective unit dosage forms.

In some embodiments, a dry powder composition is micronized forinhalation to the lungs. See for example, U.S. Patent Applicationpublication 2016/0263257, expressly incorporated herein by reference inits entirety, and in particular regarding the dry powder cromolynformulations described therein. In other embodiments, the dry powdercomposition further comprises at least one excipient. In certainembodiments, the at least one excipient comprises Lactose monohydrateand/or Magnesium stearate.

In certain embodiments, the compounds may be administered in doses thattreat the particular indication. In particular, the dose is specificallytailored to lead to blood, brain, and CSF concentrations that allow thedrugs to act as M1-to-M2 modifiers. Such doses may include from about 1mg to about 1000 mg per day.

The dosage of the active agents will generally be dependent upon anumber of factors, including the pharmacodynamic characteristics of thecompound, mode and route of administration of the compound, the healthof the patient being treated, the extent of treatment desired, thenature and kind of concurrent therapy, if any, and the frequency oftreatment and the nature of the effect desired. In general, dosageranges of the compound often range from about 0.001 to about 250 mg/kgbody weight per day. For a normal adult having a body weight of about 70kg, a dosage may range from about 0.1 to about 25 mg/kg body weight.However, some variability in this general dosage range may be requireddepending on the age and weight of the subject being treated, theintended route of administration, the particular agent beingadministered, and the like. Importantly, the determination of dosageranges and optimal dosages for a particular mammal is also well withinthe ability of one of ordinary skill in the art having the benefit ofthe instant disclosure.

Dosages for compounds may be as low as 5 ng/d. In certain embodiments,about 10 ng/day, about 15 ng/day, about 20 ng/day, about 25 ng/day,about 30 ng/day, about 35 ng/day, about 40 ng/day, about 45 ng/day,about 50 ng/day, about 60 ng/day, about 70 ng/day, about 80 ng/day,about 90 ng/day, about 100 ng/day, about 200 ng/day, about 300 ng/day,about 400 ng/day, about 500 ng/day, about 600 ng/day, about 700 ng/day,about 800 ng/day, about 900 ng/day, about 1 μg/day, about 2 μg/day,about 3 μg/day, about 4 μg/day, about 5 μg/day, about 10 μg/day, about15 μg/day, about 20 μg/day, about 30 μg/day, about 40 μg/day, about 50μg/day, about 60 μg/day, about 70 μg/day, about 80 μg/day, about 90μg/day, about 100 μg/day, about 200 μg/day, about 300 μg/day, about 400μg/day about 500 μg/day, about 600 μg/day, about 700 μg/day, about 800μg/day, about 900 μg/day, about 1 mg/day, about 2 mg/day, about 3mg/day, about 4 mg/day, about 5 mg/day, about 10 mg/day, about 15mg/day, about 20 mg/day, about 30 mg/day, about 40 mg/day or about 50mg/day of the compound is administered.

Dosage ranges for active agents may be from 5 ng/d to 100mg/day. Incertain embodiments, dosage ranges for active agents may be from about 5ng/day to about 10 ng/day, about 15 ng/day, about 20 ng/day, about 25ng/day, about 30 ng/day, about 35 ng/day, about 40 ng/day, about 45ng/day, about 50 ng/day, about 60 ng/day, about 70 ng/day, about 80ng/day, about 90 ng/day, about 100 ng/day, about 200 ng/day, about 300ng/day, about 400 ng/day, about 500 ng/day, about 600 ng/day, about 700ng/day, about 800 ng/day, or about 900 ng/day. In certain embodiments,dosage ranges for compounds may be from about 1 μg/day to about 2μg/day, about 3m/day, about 4 μg/day, about 5 μg/day, about 10 μg/day,about 15 μg/day, about 20 μg/day, about 30 μg/day, about 40 μg/day,about 50 μg/day, about 60 μg/day, about 70 μg/day, about 80 μg/day,about 90 μg/day, about 100 μg/day, about 200 μg/day, about 300 μg/day,about 400 μg/day about 500 μg/day, about 600 μg/day, about 700 μg/day,about 800 μg/day, or about 900 μg/day. In certain embodiments, dosageranges for active agents may be from about 1mg/day to about 2 mg/day,about 3 mg/day, about 4 mg/day, about 5 mg/day, about 10 mg/day, about15 mg/day, about 20 mg/day, about 30 mg/day, about 40 mg/day, about 50mg/day, about 60 mg/day, about 70 mg/day, about 80 mg/day, about 90mg/day, about 100 mg/day, about 200 mg/day, about 300 mg/day, about 400mg/day, about 500 mg/day, about 600 mg/day, about 700 mg/day, about 800mg/day, or about 900 mg/day.

In certain embodiments, the compounds are administered in pM or nMconcentrations. In certain embodiments, the compounds are administeredin about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 20 pM, about30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM,about 90 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM,about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM,about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM,about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 20 nM, about 30nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM,about 90 nM, about 100 nM, about 300 nM, about 400 nM, about 500 nM,about 600 nM, about 700 nM, about 800 nM, or about 900 nM,concentrations.

In certain embodiments, the dosage form is a solid dosage form, and thesize of the compound in the dosage form is important. In certainembodiments, the compound is less than about 3 μm, less than about 2 μm,or less than about 1 μm in diameter. In certain embodiments, the activeagent is about 0.1 μm to about 3.0 μm in diameter. In certainembodiments, the active agent is from about 0.5 μm to about 1.5 μm indiameter. In certain embodiments, the active agent is about 0.2 μm,about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm,about 0.8 μm, about 0.9 μm, about 1.0 μm, about 1.1 μm, about 1.2 μm,about 1.3 μm, about 1.4 μm, or about 1.5 μm in diameter.

For example, a formulation intended for oral administration to humansmay contain from about 0.1 mg to about 5 g of the active agent (orcompound) compounded with an appropriate and convenient carrier materialvarying from about 5% to about 95% of the total composition. Unitdosages will generally contain between about 0.5 mg to about 1500 mg ofthe active agent. The dosage may be about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg,6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg,17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg 25 mg, 26 mg, 27mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg 35 mg, 36 mg, 37 mg,38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48mg, 49 mg, 50 mg, 55 mg, 60 mg, 65, mg, 70 mg, 75 mg, 80 mg, 85 mg, 90mg, 95 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or100 mg, etc., up to about 1500 mg of the compound.

In certain embodiments, the invention relates to combination of twoactive agents. In certain embodiments, it may be advantageous for thepharmaceutical combination to be comprised of a relatively large amountof the first component compared to a second component. In certaininstances, the ratio of the first active agent to the second activeagent is about: 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1,120:1, 110:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1,15:1, 10:1, 9:1, 8:1, 7:1, 6:1, or 5:1. It further may be preferable tohave a more equal distribution of pharmaceutical agents. In certaininstances, the ratio of the first active agent to the second activeagent is about: 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, or 1:4. It may also beadvantageous for the pharmaceutical combination to have a relativelylarge amount of the second component compared to the first component. Incertain instances, the ratio of the second active agent to the firstactive agent is about 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1,130:1, 120:1, 110:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1,20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, or 5:1. A composition comprisingany of the above identified combinations of the first therapeutic agentand second therapeutic agent may be administered in divided doses about1, 2, 3, 4, 5, 6, or more times per day or in a form that will provide arate of release effective to attain the desired results. The dosage formmay contain both the first and second active agents. The dosage form maybe administered one time per day if it contains both the first andsecond active agents.

For example, a formulation intended for oral administration to humansmay contain from about 0.1 mg to about 5 g of the first therapeuticagent and about 0.1 to about 5 g of the second therapeutic agent, bothof which are compounded with an appropriate and convenient about ofcarrier material varying from about 5% to about 95% of the totalcomposition. Unit dosages will generally contain between about 0.5 mg toabout 1500 mg of the first therapeutic agent and 0.5 mg to 1500 mg ofthe second therapeutic agent. The dosage may be about: 25 mg, 50 mg, 100mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 100 mg, etc., upto about 1500 mg of the first therapeutic agent. The dosage may beabout: 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800mg, or 100 mg, etc., up to about 1500 mg of the second therapeuticagent.

In certain embodiments, the inventions relates to a method of treating aAlzheimer's disease comprising administering by inhalation a micronized,dry powder comprising about 1 mg to 100 mg of Cromolyn Disodium per dayto a patient in need thereof.

EXAMPLES Example 1

Our studies in PS1/PSS animal model showed that Cromolyn sodium impactedthe interaction of microglial cells with amyloid deposits and eventuallyaffected Aβ clearance by microglia. We first performed a doubleimmunostaining between Aβ and the microglial marker Iba1 in brainsections of mice treated with PBS or the highest dose of Cromolyn sodium(3.15mg/kg). A systematic analysis of the overlap between both stainingsrevealed that animals that received Cromolyn Sodium showed a higherpercentage of Iba1 immunoreactivity overlapping with amyloid (FIG. 1B),which may indicate a modest increased recruitment of microglia aroundplaques induced by the compound.

To go further in our understanding of these mechanisms, and consideringthat evaluating change in microglial function is challenging in vivo, weused an additional in vitro system of Aβ microglial uptake. SyntheticAβ₄₀ and Aβ₄₂ peptides were applied to microglia in culture in thepresence or absence of Cromolyn Sodium.

After 16 hours of incubation, we observed a dose dependent decrease ofAβ₄₀ and Aβ₄₂ levels in presence of Cromolyn Sodium, indicating that theimpact of Cromolyn Sodium on Aβ aggregation mechanisms may promote Aβclearance by microglial uptake (FIG. 1C). The combination of those invivo and in vitro results may suggest that, in addition to inhibitingfibrillization, Cromolyn Sodium affected microglial activation and Aβclearance.

Cromolyn Sodium does not affect the levels of Aβ in the plasma butpromotes microglial Aβ clearance. FIG. 1A illustrates the quantificationof the plasmatic levels of Aβ_(x-40) and Aβ_(x-42) one week aftertreatment with PBS or escalating doses of Cromolyn Sodium (n=3-5mice/group). FIG. 1B illustrates representative images of localizationof amyloid deposits (6E10) and microglia (Iba1) in mice treated withCromolyn Sodium (3.15mg/kg) or PBS daily for seven days. The percentageof amyloid occupied by Iba1 positive processes was calculated for eachdeposit and showed an increased overlap between Aβ and Iba1 aftertreatment with Cromolyn Sodium (n=3 mice for PBS and n=5 mice forCromolyn Sodium). Between 20 to 20 plaques were evaluated for eachanimal). Scale bar=10 μm. FIG. 1C illustrates the effect of CromolynSodium on microglial Aβ uptake in vitro. Microglial cells were culturedand incubated with 50 nM of synthetic Aβ₄₀ or Aβ42 and 0, 10 nM, 10 μMor 1 mM of Cromolyn Sodium for 16 hours. After the incubation, theconcentrations of Aβ_(x-40) (FIG. 1C left) Aβ_(x-42) (FIG. 1C, right) inmedia were measured using Aβ ELISA and normalized with microglia cellnumber and according to the PBS control condition. (n=3 experiments; *,P<0.05, **, P<0.01)

Example 2

In other animal studies of microglia activation and M1, M2 conversionshowed that cromolyn is the only of many drugs tested that effected thischange and exhibited phagocytic activity. FIG. 2 illustratesrepresentative plaques of all the plaques and the microglial cellssurrounding those deposits in Tg-2576 mice of the study. An imageanalysis looking at the percentage of Iba-1 positive processescolocalizing with the amyloid staining versus the total amount of Iba-1signal surrounding the plaque demonstrated that there was moreIba-1/Amyloid colocalization when the mice were treated with CromolynSodium as opposed to any other groups. This result correlates with ourresults in Example 1 and our in vitro data.

Cromolyn, but not ibuprofen promotes microglial Aβ₄₂ uptake, theircombination improved uptake over either ibuprofen or cromolyn alone. BV2microglial cell cultures were treated with cromolyn and/or ibuprofen (10μM, 100 μM, 1 mM) for 16 hours. Afterwards, cells were incubated withsoluble Aβ₄₂ and the compounds for 3 hours. After incubation, cells werecollected for ELISA analysis. BV2 microglial cells treated with cromolyn(100 μM, 1 mM), and with cromolyn and ibuprofen (100 μM, 1 mM for eachcompound) exhibit increased Aβ₄₂ uptake levels relative to BV2 microgliatreated with the vehicle. Results were derived from three independentexperiments; **p<0.01, ***p<0.001, one-way ANOVA, Tukey's test). Dataare represented as mean±SEM. FIG. 3 graphically illustrates the resultsof BV2 microglial cells treated with cromolyn, and with cromolyn andibuprofen exhibit increased Aβ₄₂ uptake levels relative to BV2 microgliatreated with the vehicle.

Example 3: Compound Synthesis5,5′-[(2-Hydroxy-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-carboxylicacid diethyl ester

A suspension of cromolyn sodium salt (1.0 g, 2 mmol) in EtOH (100 mL)and con. HCl (1 mL) was heated in a sealed reactor tube for 24 h at 100°C. The white solid was dissolved to give a clear colorless solutionwhile hot. It was allowed to cool to room temperature and NaHCO₃ (1.0 g)was added. After stirring for 30 min at 25° C., solvent was removed byroto-evaporation. Chromatography on silica gel of the crude materialusing 5:95 methanol/methylene chloride yielded the diethyl ester (0.8 g,76% yield); mp 154-156° C.; ¹H NMR (CDCl₃, 300 MHz) δ 1.42 (t, 3H, J=7.1Hz, CH₃), 2.73 (br s, 1H, OH), 4.44 (q, 4H, J=7.1 Hz, 2OCH₂CH₃),4.32-4.59 (m, 5H, CHOH, 2OCH₂), 6.80 (s, 2H, 2 vinyl-H), 6.99 (d, 2H,J=8.24 Hz, 2Aro-H), 7.12 (d, 2H, J=8.24 Hz, 2Aro-H), 7.17 (d, 2H, J=8.24Hz, 2Aro-H), 7.71 (t, 2H, J=8.24 2Aro-H).

5,5′-[(2-Fluoro-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-carboxylicacid diethyl ester

3-Bis(4-methylbezenesulfonate)-2-fluoropropanediol

A solution of 1,3-bis(4-methylbezenesulfonate propanetriol (2.7 g, 6.78mmol) in methylene chloride (20 mL) at 0-5° C. was treated with DAST(2.18 g, 13.6 mmol). The mixture was stirred at 0-5° C. for 30 thenallowed to warm to 25° C. and stirred for 16 hr. The mixture was pouredinto a sat'd sodium bicarbonate solution (30 mL) and layers separated.The methylene chloride layer dried (sodium sulfate). After solventremoval, the crude material was chromatographed on silica gel (methylenechloride) to yield 0.82 g (30%) of a solid; mp 99-102° C.; ¹H NMR(CDCl₃), δ 2.5 (s, 6H, CH₃), 4.15 (dd, 4H, J=12.3, 4.6 Hz, CH₂, 4.8 (dq,1H, J=47, 4.6, CHF), 7.45 (d, 4H, J=8.1Hz, Aro-H), 7.75 (d, 4H, J=8.4Hz, Aro-H).

5,5′-(2-fluoropropane-1,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylicacid)

1,3-Bis(2-acetyl-3-hydroxyphenoxy)-2-fluoropropane

A mixture of 3-bis(4-methylbezenesulfonate)-2-fluoropropanediol (1.0,2.5 mmol), 2,6-dihydroxyacetophenone (0.76 g, 5.0 mmol) and potassiumcarbonate (0.69 g) in acetonitrile (40 mL) was heated under reflux for16 hr. The mixture was filtered and the filtrate was evaporated. Thecrude material was chromatographed on silica gel (acetonitrile/methylenechloride 5:95) to yield 0.57 g (40%) of product; mp 162-165° C.; ¹H NMR(d6-DMSO), δ 2.5 (s, 6H, 2CH₃), 4.38 (m, 4H, 2CH₂), 5.22 (br d 1H, J=49Hz, CHF), 6.45 (m, 4H, 4Aro-H), 7.28 (t, 2H, J=4.55 Hz, 2Aro-H).

5,5′-[(2-Fluoro-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-carboxylicacid diethyl ester

A mixture of 1,3-bis(2-acety-3-hydroxyphenoxy)-2-fluoropropane (200 mg,0.52 mmol) and ethyl oxalate (2 mL) was added to a solution of sodiumethoxide (87 mg Na) in ethanol (10 mL) and benzene (10 mL). The mixturewas heated at reflux for 16 hr, cooled and diluted with ether (50 mL).The precipitated sodium salt was filtered, washed with ether and dried.It was then dissolved in water and acidified with 10% HCl to obtain asticky solid. The solid was refluxed in ethanol (20 mL) with a catalyticamount of 36% HCL for 1 hr. The mixture was poured into 50 mL of waterand extracted twice with methylene chloride (50 mL). The extracts werecombined and dried. After solvent removal, the crude material waschromatographed on silica gel (acetonitrile/methylene chloride 10:90) toyield 0.12 g (45%) of product; mp 166-170° C.; ¹H NMR (CDCl₃), δ 1.42(t, 6H, J=7.14 Hz, 2CH₃), 4.58 (q, 4H, J=7.14 Hz 2CH₂), 4.65 (m, 4H,2CH₂), 5.35 (dq, 1H, J=46 Hz, J=4.4 HZ, CHF), 6.90 (s, 2H, vinyl-H),6.95 (d, 2H, J=8.24 Hz, 2Aro-H), 7.13(d, 2H, J=8.24 Hz, 2Aro-H),7.17 (d,2H, J=8.24 Hz, 2Aro-H) 7.6 (t, 2H, J=8.24 2Aro-H).

5,5′-[(2-Fluoro-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-carboxylicacid

A suspension of5,5′-[(2-fluoro-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-carboxylicacid diethyl ester (100 mg, 0.19 mmol) in methanol (20 mL) and 1 Msodium hydroxide (2 mL) was heated at 80° C. for 1 hr. The solution wasacidified with 10% HCl and volatiles were removed. A solution ofmethanol/methylene chloride (50:50) was added to the solid and themixture was filtered. Evaporation afforded 76 mg (85%) of product; ¹HNMR (d6-DMSO), δ 4.65 (m, 4H, 2CH₂), 5.32 (br d, 1H, J=46 Hz, CHF), 6.80(s, 2H, 2vinyl-H), 7.2 (d, 2H, J=8.24 Hz, 2Aro-H), 7.71 (t, 2H, J=8.242Aro-H).

5,5′-[(2-Hydroxy-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-ethanol

To a suspension of5,5′-(2-hydroxytrimethylenedioxy)bis(4-oxochromene-2-carboxylic acid)diethyl ester (1.0 g, 1.86 mmol) in methanol (60 ml) and methylenechloride (40 mL) NaBH₄ (0.14 g, 3.72 mmol) was added in portions over a1 h period. The mixture was stirred at 25° C. until it was clear(approx. 5 h) at which time the solution was quenched by dropwiseaddition of 1M HCl until acidic. Solvent was evaporated and the residuewas extracted with methylene chloride. The combined organic extractswere washed with water and dried over anhydrous sodium sulfate. Afterevaporation, the residue was purified by column chromatography (5:95methanol/methylene chloride) to yield 0.5 g (50%) of the triol; ¹H NMR(DMSO-d₆, 300 MHz) δ 2.73 (s, 3H, OH), 4.25-4.36 (m, 9H, 2OCH₂, CH—O),6.13 (s, 2H, 2 vinyl H), 7.04 (d, 2H, J=8.4 Hz, aromatic H), 7.07 (d,2H, J=8.4 Hz, aromatic H), 7.63 (t, 2H, J=8.2 Hz, aromatic H).

5,5′-[(2-Fluoro-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-ethanol

The above procedure for5,5′-(2-hydroxytrimethylenedioxy)bis(4-oxochromene-2-ethanol) was used.¹H NMR (DMSO-d₆, 300 MHz) δ 2.73 (s, 3H, OH), 4.25-4.36 (m, 8H, 2OCH₂,CH—O), 5.35 (br d, 1H, J=46 Hz, CHF), 6.13 (s, 2H, 2 vinyl H), 7.04 (d,2H, J=8.4 Hz, aromatic H), 7.07 (d, 2H, J=8.4 Hz, aromatic H), 7.63 (t,2H, J=8.2 Hz, aromatic H).

5,5′-[(2-Hydroxy-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-carboxylicacid bis[(2,2-dimethyl-1-oxopropoxy)methyl] ester

To a stirred solution of cromolyn diacid (1.0 g, 2.7 mm) in 20 mL of DMFwas added diisopropylamine (0.7 mL) and 1.0 g (6.5 mmol)chloromethylpivalate. The reaction mixture was stirred at 60° C. for 4hr, water was added and the mixture was extracted with separated, dried(MgSO₄) and the solvent removed in vacuo. The solvent was removed andthe residue was chromatographed on silica 4% methanol in methylenechloride to give 1.2 g (65%) of the pivalate compound; mp 135-140° C.;H¹ NMR (CDCl), δ 1.24 (s, 18 H, CH₃), 4.36 (m, 2 H, OCH₂), 4.49 (m, 1 H,CHOH), 4.51 (m, 2H, OCH₂),), 6.00 (s, 4H, CH—O—CO), 6.98 (m, 4H,2vinyl-H, 2Aro-H), 7.13 (d, 2H, J=8.24 Hz, 2Aro-H), 7.61 (t, 2H, J=8.242Aro-H).

5,5′-[(2-Fluoro-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-carboxylicacid bis[(2,2-dimethyl-1-oxopropoxy)methyl] ester

To a stirred solution of5,5′-[(2-fluoro-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-carboxylicacid (1.0 g, 2.1 mmol) in 20 mL of DMF was added iisopropylamine (0.7mL) and 1.0 g (6.5 mmol) chloromethylpivalate. The reaction mixture wasstirred at 60° C. for 4 hr, water was added and the mixture wasextracted with separated, dried (MgSO₄) and the solvent removed invacuo. The solvent was removed and the residue was chromatographed onsilica using 2% methanol in methylene chloride to give 1.0 g (70%) ofthe pivalate compound; mp 130-133° C.; δ 1.21 (s, 18 H, CH₃), 4.36 (m, 4m, 2OCH₂), 4.49 (br d, 1H, J=46 Hz, CHF), 6.00 (s, 4H, CH—O—CO), 6.98(m, 4H, 2vinyl-H, 2Aro-H), 7.13 (d, 2H, J=8.24 Hz, 2Aro-H), 7.61 (t, 2H,J=8.24 2Aro-H).

Triacetate of5,5′-[(2-hydroxy-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-ethanol

Acetic anhydride (0.5 g, 4.6 mmol)) was slowly added to a mixture of5,5′-[(2-hydroxy-1,3-propanediyl)bis(oxy)]bis[4-oxo-4H-1-benzopyran-2-ethanol(0.5 g, 1.14 mmol) in pyridine (20 mL) cooled to 0-5° C. The mixture wasstirred for 3 hr at 0-5° C. and then allowed to warm to roomtemperature. TLC indicted the reaction was complete. Methylene chloridewas added and the mixture was washed with 10% HCl until the aqueousphase was acidic. The methylene chloride layer was dried over anhydroussodium sulfate and solvent was evaporated. Chromatography on silicausing 3% methanol in methylene chloride gave 0.45 g (72%) of thetriacetate compound; mp 122-125° C.; H¹ NMR (CDCl₃), δ 2.16 (s, 9 H,CH₃), 4.58 (m, 2 H, CH₂OH), 4.66 (m, 2H, CH₂OH), 4.94 (s, 4 H, CH₂OH),5.66 (m, 1 H, CHOH), 6.15 (s, 2H, 2vinyl-H), 6.94 (d, 2H, 2Aro-H), 6.97(d, 2H, J=8.24 Hz, 2Aro-H), 7.52 (t, 2H, J=8.24, 2Aro-H).

Example 4: Aβ Aggregation Inhibition Assay

Experimental design. 3-month old Tg2576 mice were acclimatized for 2months, and then randomly assigned to different treatment groups. Theyincluded the control group (n=10) with vehicle treatment, the cromolynlow dose group and cromolyn high dose group. The treatments wereconducted through IP injection with PBS based on 0.1 mL/30 g bodyweight, 3 times per week for 3 additional months. All mice weresacrificed at 8-month old. Tissues were harvested and processed forpostmortem analysis.

Synthetic Aaβ₄₂ in final 5 uM was incubated with 10, 100, 1,000 nM oftest compounds for 1 hour. The aggregation was initiated with heparin at0.5 mg/ml in final concentration. The assay buffer consisted of 125 mMNaCl, 2.5 mM KCl, 1 mM MgCl₂, 1.25 mM Na₂H₂PO₄, 2 mM CaCl₂, 25 mMGlucose, and NaHCO₃ to adjust pH to 7.4. The assay buffer was used as acontrol. The aggregation was measured by intensity of Thioflavin Tbinding, which was detected by fluorescent excitation/emission at 450nm/480 nm (Spectra Max M3 plate reader, Molecular Devices) in a kineticmode. Aggregation was recorded as the kinetics was calculated as Vmax bythe plate reader's software. The assay was performed in triplicate andexpressed as standard mean±SD. Blue dotted line indicate the assaybuffer control. FIG. 4 illustrates the results of the assay.

Example 5

Cromolyn significantly affected the levels of brain TBS soluble Aβ andthe ratios of Aβ (42:40). A-B. MSD (mess scale to measure Aβ 42,40,and38) Aβ analyses were apply to brain TBS soluble samples. Differences inthe Aβ levels and the ratios of Aβ (42:40) comparing the varioustreatment groups were analyzed. *p<0.05; **p<0.01, ***p<0.001, one-wayANOVA, Tukey's test; mean±SEM show that cromolyn and ibuprofencombination for the low and high dose higher relative level of Aβ 42/40and ah higher Aβ 38 that is not implicated in plaque formation. FIG. 5graphically illustrates the results of a one-way of the differences inthe Aβ levels and the ratios of Aβ (42:40).

Example 6—Effect of Cromolyn Sodium on Aβ42 Uptake in Microglial Cells

Confocal microscopy and ELISA assays were used to assess the effect ofcromolyn and its derivative compounds on Aβ42 uptake in microglialcells. The BV2 microglial cell line, which was previously found toefficiently take up and degrade exogenously-added Aβ42, was used (Jiang,Q., et al. (2008) Neuron 58, 681-693; Mandrekar et al., 2009 J.Neurosci. 29, 4252-4262). The compounds were tested in naïve BV2microglial cells to investigate whether they modulate Aβ uptake. Theeffect of compounds in BV2 cells stably expressing full-length humanCD33 (BV2-CD33^(WT)) was assessed to explore whether they reverseCD33-mediated inhibition of Aβ uptake (Griciuc et al., 2013 Neuron 78,631-643).

The compound numbers, molecular weight and concentration of the stocksolutions are summarized in Table 1. Cromolyn derivatives, C3 and C4,displayed lower solubility in DMSO in comparison to C1, C2, C5, C6, C7and C8. Therefore, a 25 mM stock solutions for all the compounds exceptfor C3 and C4 were prepared. The stock solutions for C3 and C4 wereprepared at 5 mM and 7.5 mM, respectively. C1 is the parentcompound—cromolyn disodium.

TABLE 1 Summary of compounds tested in microglial cells Compound StockSolution Number Compound Name (mM) C1 Cromolyn Disodium 25 C2 F-CromolynDiacid 25 C3 ET-Cromolyn 5 C4 F-ET-Cromolyn 7.5 C5 Triol-Cromolyn 25 C6F-Triol-Cromolyn 25 C7 Ac-Triol-Cromolyn 25 C8 POM-Cromolyn 25

To investigate the effect of cromolyn sodium on Aβ42 uptake inmicroglial cells, naïve BV2 cells were treated with DMSO (control) orcromolyn at 500 μM for 16 hours. Afterwards, cells were washed with PBSand treated with DMSO or cromolyn in the presence of thefluorescently-tagged Aβ42 peptide (400 nM, red) for 2 hours. At the endof the treatment, the cells were washed and labeled them with a plasmamembrane dye (green). Using confocal microscopy and the fluorescencesignal in the red channel, the levels of intracellular Aβ42 peptide werequantified. All the quantifications were performed by a blind observerwith the ImageJ software. Remarkably, cromolyn sodium led to increaseduptake of Aβ42 in naïve BV2 microglial cells (FIG. 6A-FIG. 6D).

Furthermore, whether cromolyn sodium modulates Aβ42 uptake in naïve BV2microglial cells was determined by using the ELISA assay. Additionally,whether cromolyn sodium leads to increased Aβ42 uptake levels in BV2cells stably expressing full-length human CD33 (BV2-CD33^(WT)) wasdetermined. To this purpose, both naïve BV2 and BV2-CD33^(WT) cell lineswere treated with DMSO (control) or cromolyn at different concentrationsfor 16 hours. Then, the cells were washed with PBS and treated with DMSOor cromolyn and soluble untagged Aβ42 peptide (400 nM) for 2 hours. Thecollected cell lysates were analyzed for Aβ42 uptake levels using theAβ42-specific ELISA kit from Wako. The ELISA results were normalized tothe protein concentration levels that were previously quantified usingthe BCA assay.

Cromolyn sodium led to increased Aβ42 uptake at 100 μM and 1 mM in naïveBV2 microglial cells (FIG. 7A) and thus, confirmed theimmunofluorescence results by ELISA assay. Cromolyn sodium also led toincreased levels of internalized Aβ42 at 10 μM and 500 μM inBV2-CD33^(WT) cells (FIG. 7B, ELISA assay) and reversed CD33-mediatedinhibition of Aβ42 uptake in microglial cells. In conclusion, treatmentwith cromolyn sodium showed a dose-dependent effect in modulating Aβ42uptake levels in naïve BV2 and BV2-CD33^(WT) cell lines.

Example 7—Effect of Cromolyn Derivatives on Aβ42 Uptake in MicroglialCells

To investigate the effect of cromolyn derivatives on Aβ42 uptake inmicroglia, naïve BV2 or BV2-CD33^(WT) cells were plated in proliferatingmedia. On the following day, cells were treated with DMSO (control) orthe compounds at different concentrations in proliferating media for 3hours. C1, C2, C5, C6, C7 and C8 were tested at 10, 50, 100 and 150 μM,while C3 and C4 were assessed at 5, 25, 50 and 75 μM due to solubilitylimit in DMSO. Afterwards, cells were washed with PBS and treated withDMSO or compounds in the presence of the untagged Aβ42 peptide (400 nM)in DMEM media for 2 hours. Compound toxicity was assessed in the mediacollected at the end of the treatment with CytoTox-ONE™ lactatedehydrogenase (LDH) assay. The remaining cells in the plate were washedwith cold PBS and lysed with RIPA buffer supplemented with protease andphosphatase inhibitors. Protein concentrations in the lysatesupernatants were determined using the Pierce™ BCA protein assay kit and2-3 μg/well of total protein from each lysate was analyzed for Aβ42uptake using the Aβ42 ELISA kit from Wako. Toxic compound concentrationswere excluded from further studies.

To investigate whether cromolyn derivatives induce cytotoxicity athigher doses, naïve BV2 microglial cells were incubated with DMSO(vehicle) or cromolyn derivatives at different concentrations for 3hours. The cells were then washed and incubated with DMSO or compoundsand soluble untagged Aβ42 for additional 2 hours. Afterwards, the cellmedia was collected and measured LDH released by the damaged cells toidentify the compounds that induce cytolysis. The LDH assay showed thatthe cromolyn derivative C8 is the only compound showing toxicity whentested at 100 and 150 μM (FIG. 8). Therefore, 100 and 150 μMconcentrations for C8 were excluded from the Aβ42 uptake assays.

Example 8—Modulation of Aβ42 Uptake in Microglial Cells by CromolynDerivatives

To test whether cromolyn derivatives modulate Aβ42 uptake, naïve BV2microglial cells were treated with DMSO (control) or cromolyn derivativecompounds at different concentrations for 3 hours. Afterwards, the cellswere washed and treated with DMSO or compounds in the presence ofuntagged Aβ42 peptide for 2 hours. At the end of the treatment, the celllysates were collected. The analysis for intracellular Aβ42 levels isperformed using an Aβ42-specific ELISA kit. The parent compound C1(cromolyn sodium) led to a modest increase of Aβ42 uptake at 100 and 150μM in BV2 cells. The Cl aliquot received with the other cromolynderivatives displayed lower solubility in DMSO than the C1 aliquot thatwas sent to us the first time (without the cromolyn derivatives).Interestingly, the compound C6 led to a robust inhibition of Aβ42 uptakein BV2 microglial cells. Remarkably, the cromolyn derivative C4 led toan increased uptake of Aβ42 peptide at 75 μM in naïve BV2 microglialcells (FIG. 9).

Further, whether cromolyn derivatives impact Aβ42 uptake and clearancein BV2-CD33^(WT) cells was determined by two independent sets ofexperiments. BV2-CD33^(WT) cells were treated with DMSO (control) orcromolyn derivatives at different concentrations ranging between 5 and150 μM.

In the first set of experiments, the cromolyn derivatives C1 and C3-8were tested. The compound C2 was tested with other cromolyn derivativesin the second set of experiments. Treatment with the compound C4 at 75μM resulted in a two-fold increase in Aβ42 uptake in comparison to DMSOtreatment and displayed a dose-dependent effect at 50 μM (FIG. 10).Using the GraphPad Prism 7 Software, the IC₅₀ for C4 was 54.7 μM inBV2-CD33^(WT) cells. The compound C6 exhibits a dose-dependent effect inmediating inhibition of Aβ42 uptake in BV2-CD33^(WT) cells when comparedto DMSO treatment.

In the second set of experiments, the cromolyn derivatives C1, C2, andC4-7 in BV2-CD33^(WT) cells was tested. These results confirmed priorresults that the compound C4 was the most effective in increasing theAβ42 uptake at 75 μM and displayed a dose-dependent effect at lowerconcentrations when compared to DMSO treatment (FIG. 11). Thus, theseresults suggest that the compound C4 led to increased Aβ42 uptake levelsin BV2-CD33^(WT) cells and reversed the CD33-mediated inhibition of Aβuptake and clearance (FIGS. 10 and 11).

These results suggest that the cromolyn derivative C4 induced microglialuptake and clearance of Aβ42 and enhanced skewing of microglial cellsfrom the neurotoxic/pro-inflammatory towardsneuroprotective/pro-phagocytic activation phenotype.

1. A method of treating a neuron inflammation condition in a patient inneed thereof comprising administering to the patient a therapeuticallyeffective amount of at least one compound having the following formula:

provided the compound is not cromolyn disodium,

when the neuron inflammation condition is Alzheimer's Disease (AD). 2.The method according to claim 1, wherein the compound has the followingformula


3. The method of claim 1, wherein the neuron inflammation condition isamyotrophic lateral scerlosis (ALS).
 4. The method of claim 1, whereinthe neuron inflammation condition is AD.
 5. The method of claim 1,wherein the neuron inflammation is Huntington's Disease.
 6. The methodof claim 1, wherein the neuron inflammation is Parkinson's disease (PD).7. The method of claim 1, wherein the neuron inflammation condition isischemic stroke.
 8. The method of claim 1, wherein the neuroninflammation condition is associated with prion disease. 9-13.(canceled)
 14. The method of claim 3, further comprisingco-administering a second compound selected from CD4+; siRNA; miRNA thatameliorates ALS; glial morphology modifier; SOD1 control; and Riluzole.15. The method of claim 3, further comprising co-administering a secondcompound selected from an anti-aggregation drug and a targeting drug forAD.
 16. The method of claim 1, wherein the neuron inflammation conditionis AD, further comprising co-administering a second compound selectedfrom an antibody targeting drug that ameliorates AD.
 17. The method ofclaim 1, wherein the neuron inflammation condition is AD, furthercomprising co-administering a second compound selected from ananti-inflammatory targeting drug that ameliorates AD.
 18. The method ofclaim 1, wherein the neuron inflammation condition is AD, furthercomprising co-administering a second compound selected from a tautargeting drug that ameliorates AD.
 19. The method of claim 3, furthercomprising co-administering a second compound selected from an antibodytargeting drug that ameliorates ALS.
 20. The method of claim 3, furthercomprising co-administering a second compound selected from ananti-inflammatory targeting drug that ameliorates ALS.
 21. The method ofclaim 1, further comprising co-administering a second compound selectedfrom a targeting drug that ameliorates neurodegeneration associated withamyloidosis or tauopathies.
 22. The method of claim 6, furthercomprising co-administering a second compound selected from an alphasynuclein targeting drug that ameliorates PD and a Parkinson's targetingdrug that ameliorates PD.
 23. The method of claim 3, wherein thecompound is cromolyn disodium.
 24. The method of claim 5, wherein thecompound is cromolyn disodium.
 25. The method of claim 6, wherein thecompound is cromolyn disodium.